Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces
- The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of α-subunits. The N termini of α-subunits might partly occlude the entrance channel in α-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.
Author: | Andreas ThessORCiD, Silke HutschenreiterGND, Matthias HofmannGND, Robert TampéORCiDGND, Wolfgang BaumeisterORCiDGND, Reinhard Guckenberger |
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URN: | urn:nbn:de:hebis:30:3-759984 |
DOI: | https://doi.org/10.1074/jbc.M202145200 |
ISSN: | 0021-9258 |
Parent Title (English): | Journal of biological chemistry |
Publisher: | American Society for Biochemistry and Molecular Biology Publications |
Place of publication: | Bethesda, Md |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2021/01/04 |
Year of first Publication: | 2002 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2023/11/09 |
Volume: | 277 |
Issue: | 39 |
Page Number: | 8 |
First Page: | 36321 |
Last Page: | 36328 |
HeBIS-PPN: | 516505823 |
Institutes: | Biochemie, Chemie und Pharmazie |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - Namensnennung 4.0 |